SFEBES2009 Poster Presentations Endocrine tumours and neoplasia (39 abstracts)
Functional characterisation of aryl hydrocarbon receptor interacting protein (AIP) promoter and silent mutations
Susana Igreja 1 , Harvinder Chahal 1 , Peter King 1 , Graeme Bolger 2 , Umasuthan Srirangalingam 1 , Leonardo Guasti 1 , Paul Chappel 1 , Maria Gueorguiev 1 , Katie Guegan 3 , Karen Stals 3 , Bernard Khoo 4 , Ajith Kumar 1 , Sian Ellard 3 , Ashley B Grossman 1 & Márta Korbonits 1
1Department of Endocrinology, Barts and the London School of Medicine, Queen Mary University of London, London, UK; 2Comprehensive Cancer Center, University of Alabama, Birmingham, Alabama, USA; 3Department of Molecular Genetics, Royal Devon and Exeter Foundation Trust, Exeter, UK; 4Department of Endocrinology, UCL Medical School, Royal Free Campus, London, UK.
AIP mutations predispose to familial isolated pituitary adenomas (FIPA) and 45 different AIP mutations have been described in the literature. Most of these mutations result in complete disruption of the C-terminal region of the AIP protein due to early stop codons. In this study we were particularly interested in the effect of AIP mutations in the promoter region (−270−−269CG & −220C) and 2 synonymous mutations (c.249G>T, p.G83= and c.807C>T, p.F269=) identified in FIPA families.
An in vitro luciferase assay was used to study promoter activity and regulation, while patient’s RNA and minigene constructs were utilised to study splicing abnormalities.
Luciferase assay studies of the dibasic promoter mutation showed reduced basal promoter activity in GH3 cells (P<0.001). Furthermore, stimulation with forskolin, db-cAMP (activators of the PKA signalling pathway) and phorbol-12-myristate-13-acetate (activator of the PKC signalling pathway) revealed that the cAMP-PKA signalling pathway positively regulates the AIP promoter. The effect of forskolin on the WT promoter was inhibited by the PKA inhibitor H89. The silent mutation c.249G>T created a new splice-site leading to a frameshift and premature stop-codon and we were able to identify the altered cDNA product from the patient’s blood cDNA sample. The c.807C>T silent mutation led to decreased AIP expression both in patient samples and in the in vitro minigene study.
In summary, we have shown that exploration of gene changes such as those in the promoter region or silent exonic mutations results in the putative involvement of AIP in our FIPA families. The functional characterisation of silent AIP sequence changes is important to identify their pathogenic role in pituitary tumourigenesis.
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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